L-asparaginase is known to have therapeutic value in the treatment of Leukemia. Till date the enzymes obtained from Escherichia coli, and Erwinia chrysanthemi are being used for the same. L-asparaginase is an amidohydrolase which catalyzes L-asparagine into L-aspartic acid and ammonia. It plays a major role in the metabolism of L-asparagine in plants, animals and microorganisms. It has been energetically studied on its actual use as an antitumor agent since John G. Kidd et al had described the inhibitory action of L-asparaginase from guinea pig sera on lymphomas in “The Journal of Experimental Medicine”, Vol. 98, pp. 565-582 (1953) and then evidenced by J. D. Broome et al. in “Nature”, Vol. 191, pp. 1, 114-1, 115 (1961). It is now well established that the inhibitory action of the enzyme is caused by the depletion/removal of circulatory L-asparagine, an essential nutrient to proliferate and survive for some tumor (leukemic) cells which are compromised in L-asparagine synthesis ability, but not for the normal cells. The administration of L-asparaginase into leukemic patients induces the selective death of the tumor cells by hydrolyzing L-asparagine, resulting in the treatment of malignant tumors.
L-asparaginase was purified and characterized from several sources, bacteria, (Escherichia coli, Erwinia carotovora), plants (Withania somnifera), fungi, (Aspergillus niger, A. oryzae) etc. Among mammals, L-asparaginase is found in more than trace amounts only in Guinea pigs (superfamily Cavioidea) and in certain New World monkeys. Of these L-asparaginases from E. coli and Er. chrysanthemi are commercially available for the treatment of leukemia. E. coli L-asparaginase II (also known as L-asparagine amidohydrolase, type EC-2, EC 3.5.1.1) is commercially available as Elspar® (Merck & Co., Inc.) and is also available from Kyowa Hakko Kogyo Co., Ltd.
The available asparaginases with potent anti-leukemic activity, upon administration to the patients resulted in a wide range of host toxicity (e.g., hepatic, renal, splenic, pancreatic dysfunction and blood coagulation) and pronounced immuno-suppression (Ohno, R. & Hersh, E. M, Immunosuppressive effects of L-asparaginase, 30 Cancer Res. 1605 (1970)). Another effect of E. coli asparaginase treatment on spleen and lymphocyte was found as a marked reduction in both the size and reactivity of the splenic germinal centers concomitant with a reduction in lymphocyte population (Distasio, J. A., et al., Alteration in spleen lymphoid populations associated with specific amino acid depletion during L-asparaginase treatment, 42 Cancer Res. 252 (1982)). Hepatic dysfunction is another important adverse clinical effect associated with traditional microbial asparaginase treatment (Schein, P. S., et al., The toxicity of E. coli asparaginase, 29 Cancer Res. 426 (1969)). The indications of asparaginase-induced hepatic dysfunction and pathology include decreased plasma levels of albumin, anti-thrombin III, cholesterol, phospholipids, and triglycerides and fatty degenerative changes, delayed bromo-sulfophthalein clearance, and increased levels of serum glutamic-oxaloacetic transaminase and alkaline phosphatase. A marked decreased in spleen lymphocytic cells of the B-cell lineage and hepatotoxic effects of currently available asparaginases may be a result of depletion of both asparagine and glutamine hydrolysed by asparaginase. E. coli asparaginase has been shown to possess a 2% of glutaminase activity resulting in the observed glutamine deprivation and asparaginase-induced clinical toxicity (Spiers, A. D. S., et al., L-glutaminase/L-asparaginase: human pharmacology, toxicology, and activity in acute leukemia, 63 Cancer Treat. Rep. 1019 (1979)).
Another significant problem associated with the use of microbial asparaginases is that patients treated with E. coli and Er. carotovora asparaginases frequently develop neutralizing antibodies of the IgG and IgM immunoglobulin class (e.g., Cheung, N. & Chau, K., Antibody response to Escherichia coli L-asparaginase: Prognostic significance and clinical utility of antibody measurement, 8 Am. J. Pediatric Hematol. Oncol. 99 (1986); Howard, J. B. & Carpenter, F. H. (1972) supra), which allows an immediate rebound of serum levels of asparagine and glutamine. In an attempt to mitigate both the toxic effects and immunosensitivity associated with the therapeutic utilization of E. coli and Er. carotovora asparaginase, a covalently-modified E. coli asparaginase (PEG-asparaginase) was initially developed for use in patients who have developed a delayed-type hypersensitivity to preparations “native” of E. coli asparaginase (see Gao, S. & Zhao, G., Chemical modification of enzyme molecules to improve their characteristics, 613 Ann. NY Acad. Sci. 460 (1990)). However, subsequent studies established that the initial development of an immune response against E. coli asparaginase resulted in an 80% cross-reactivity against the PEG-asparaginase with concomitant adverse pharmacokinetic effects-neutralization of PEG-asparaginase activity and normalization of the plasma levels of L-asparagine and L-glutamine (see Avramis, V. & Periclou, I., Pharmacodynamic studies of PEG-asparaginase (PEG-ASNase) in pediatric ALL leukemia patients, Seventh International Congress on Anti-Cancer Treatment, Paris, France (1997)). The development of antibodies directed against E. coli asparaginase and the modified PEG-asparaginase in patients is associated with neutralization of the enzymatic activity of both the E. coli and PEG-asparaginases in vivo, thus potentially resulting in an adverse clinical prognosis.
Beside these, the available enzymes are unstable, having reduced half life requiring multiple dose administration, and require low storage temperature (˜2-8° C.). All these factors add to an increase in production cost and results in higher treatment cost.
For making enzyme more thermostable and specific to substrate, protein engineering attempts have been made on the available L-asparaginases (Li, L Z. et al, Enhancing the thermo-stability of Escherichia coli L-asparaginase II by substitution with pro in predicted hydrogen-bonded turn structures, Enzyme and Microbial Technology, 41 523-527 (2007), Derst C et al, Engineering the substrate specificity of Escherichia coli asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248, Protein Sci. 9 (10) 2009-17, (2000)). A reverse approach to make a thermostable L-asparaginase active at mesophilic conditions has not been attempted so far.
The present invention has been carried out in order to provide asparaginase mutants obtained from Pyrococcus furiosus which are stable in nature and are devoid in the drawbacks as enumerated above.